Teratocarcinoma Cell Line F9 Research Articles

Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC 50 ~ 1 μg/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to α6β1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Selective synthetic ligands for human nuclear retinoic acid receptors.

From a series of naphthalene and benzoic acid derivatives we have identified synthetic retinoic acid analogues exhibiting high selectivity for the nuclear retinoic acid receptors RAR alpha (Am 580), RAR beta (CD 2019) and RAR gamma (CD 437) as well as ligands sharing high affinities for all RAR subtypes (CD 367). The compounds were evaluated in two complementary screening systems: (1) binding to nuclear proteins extracted from COS-7 cells after transfection with the appropriate expression vectors, and (2) induction of plasminogen activator in the embryonic mouse teratocarcinoma cell line F9. All compounds behaved as retinoic acid agonists in the F9 test.

Adenovirus 12S E1A gene represses differentiation of F9 teratocarcinoma cells.

The F9 teratocarcinoma cell line differentiates in vitro after treatment with retinoic acid and cAMP and has been a widely used model system for the study of the molecular events that are responsible for cellular commitment and differentiation during early development. Previous experiments have suggested intriguing parallels between the control of gene expression during F9 cell differentiation and the regulation of gene expression by adenovirus E1A. Transfection of a 12S E1A-expressing plasmid into terminally differentiated, nonproliferating F9 cells generates, at high frequency, colonies of dividing cells, each of which expresses E1A. Cell lines established from these colonies proliferate in the presence of retinoic acid and have lost the fully differentiated phenotype as characterized by the absence of expression of a series of differentiation-specific genes. We conclude that expression of the viral 12S E1A gene product interferes with retinoic acid-induced F9 cell differentiation. Moreover, the results suggest that the differentiation process, as defined by markers of terminal differentiation, may not be a permanent event but can be reversed by E1A expression.

Defective Post-translational Modification of Collagen IV in a Mutant F9 Teratocarcinoma Cell Line Is Associated with Delayed Differentiation and Growth Arrest in Response to Retinoic Acid

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.

A monoclonal antibody, MA21, recognizes a surface component that is present on F9 teratocarcinoma cells and that appears vectorially on the trophectoderm of peri-implantation-stage mouse blastocysts

A monoclonal antibody (MAb) “MA21”, derived from lymphoid tissue of a multiparous mouse and selected for binding to mouse teratocarcinoma cell line F9, recognizes a surface antigen that appears on peri-implantation-stage mouse blastocysts. In indirect immunofluorescence assays, MAb MA21 does not bind to I-cell-through morula-stage embryos, nor to early, 3.5-day post-coitum (p.c.) blastocysts. When 3.5-day p.c. blastocysts are maintained 17 h in vitro and then assayed, MAb MA21 binds to a limited number of trophectoderm cells that are centered at the embryonic pole. As culture time lengthens, the number of antigen-expressing trophectoderm cells increases, forming a cap that spreads from the embryonic pole into the abembryonic region. Embryos maintained 48 h in vitro bind MAb MA21 over as much as 100% of the trophectoderm surface. MAb MA21 does not bind to the inner cell mass. When mouse pregnancy uteri are assayed by the immunoperoxidase method, MAb MA21 binds to extra-embryonic ectoderm and trophectoderm of 5-day p.c. implanted blastocysts, but does not bind to 6-day p.c. blastocysts. MAb MA21 recognizes a component with an estimated mol. wt of 44,000 from NP-40 detergent extracts of F9 cells and peri-implantation-stage mouse blastocysts. The component appears to be firmly associated with the plasma membrane; it is resistant to removal by high salt or moderate concentrations of non-ionic detergent.

Nuclear lamina heterogeneity in mammalian cells. Differential expression of the major lamins and variations in lamin B phosphorylation.

We have studied the molecular composition of the nuclear lamina in rat tissues of distinct embryological origin and the occurrence of the nuclear lamins during in vitro differentiation of the mouse F9 teratocarcinoma cell line. Immunochemical analysis demonstrated that all rat tissues contained the three major lamin forms (lamins A, B, and C) previously recognized in rat liver nuclei; however, other minor cross-reactive components were also identified in some tissues. The amount of the 67-kDa lamin B complexed with lamins A and C in the laminae of different tissues ranged from a stoichiometry of much less than 1 to approximately 1. Furthermore, it was found that F9 stem cells and their differentiated progeny express only lamin B, and Northern blotting analysis indicated that these cells fail to accumulate lamin A and C mRNA. Chemical cleavages and peptide mapping suggested that the 67-kDa lamin B form was of similar primary structure in all differentiated tissues and F9 cells. Employing antibodies with different affinities for phosphorylated and nonphosphorylated lamin B, we showed that the apparent invariance in the expression of this polypeptide is overriden by a heterogeneity produced via tissue-specific phosphorylation. Because similar differences in antibody recognition could be reproduced in vitro by phosphorylating lamin B with protein kinase A, we have concluded that the tissue-specific modifications of this protein may occur at consensus sites recognized by this enzyme. These data support the hypotheses that the lamins can form functional laminae by associating at various combinations, and that processes including differential lamin synthesis and post-translational modification can produce a steady state lamina heterogeneity.

Retinoic acid-induced differentiation of the mouse teratocarcinoma cell line F9 is accompanied by an increase in the activity of UDP-galactose: beta-D-galactosyl-alpha 1,3-galactosyltransferase.

We report here that both the mouse teratocarcinoma F9 cells and F9 cells induced to differentiate by treatment with retinoic acid contain cell surface glycoconjugates with terminal alpha-linked galactose residues, as shown by agglutination of cells with antisera to blood type B, but not to type A. In addition, both cell types contain high numbers of binding sites for Griffonia simplicifolia-I, a lectin which binds to terminal alpha-linked galactose residues, although differentiated F9 cells contain approximately 50% more binding sites/cell for this lectin. We have also confirmed that differentiation is accompanied by a decrease in the expression of the fucose-containing stage-specific embryonic antigen (SSEA)-1, as evidenced by the fact that F9 cells, but not differentiated F9 cells, are agglutinated by monoclonal antibody to this antigen. Since these results indicate that surface glycoconjugates contain terminal alpha-linked galactose residues, we assayed cell extracts for the enzyme UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase. We have found that F9 cell extracts contain this activity, and differentiation results in a significant increase in the specific activity of the enzyme, from approximately 2 nmol/mg h in F9 extracts to 7 nmol/mg h in RA/F9 extracts. It has been suggested that the loss of the SSEA-1 antigen upon differentiation of F9 cells is due to decreased activity of the enzyme GDP-Fuc:beta-D-GlcNAc-alpha 1, 3-fucosyltransferase. We therefore determined the activities of this fucosyltransferase and several other glycosyltransferases, which included UDP-GlcNAc:beta-D-Gal-beta 1,3-N-acetylglucosaminyltransferase, UDP-Gal:beta-D-GlcNAc-beta 1,4-galactosyltransferase, and GDP-Fuc:beta-D-GlcNAc-alpha 1,6-fucosyltransferase. We have found that extracts from both cell types contain these enzyme activities; differentiation, however, does not result in substantial changes in any of these activities.

A multigene family encoding several “finger” structures is present and differentially active in mammalian genomes

Mouse genomic DNA contains multiple copies of sequences homologous to the Drosophila “ Krüppel,” a member of the “gap” class of developmental control genes of the fruit fly. The most interesting aspect of the homologous region is that, like Xenopus TFIIIA, it contains multiple finger-like folded domains capable of binding to nucleic acids. We have isolated six individual phages from a mouse genomic library on the basis of their DNA homology to Krüppel finger-coding probes, and describe here the DNA sequence and expression of two such clones containing finger-like structures. Upon differentiation of mouse teratocarcinoma cell line F9 with retinoic acid and cAMP, the expression of both genes was drastically reduced, and in one instance was undetectable. Each of the several other eukaryotic DNAs analyzed contained multiple copies of homologous genes with putative finger structures, indicating the presence of a finger-containing multigene family in higher organisms.

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Cell surface structures required for B-cell activation with haptenated syngeneic cells.

The requirement for H-2-coded antigens on the cell surface of stimulator cells used for induction of B-cell responsiveness against haptenated (fluorescein isothiocyanate (FITC)) syngeneic cells was studied using H-2-less F9 teratocarcinoma cell lines. I found that FITC-labelled F9 cells, in contrast to normal spleen cells, could not induce hapten-specific antibody synthesis. The effect of treatment of stimulator cells with glutaraldehyde or trypsin before or after hapten labelling was also analysed. It was found that regardless of the order of treatment, hapten-specific antibody synthesis could not be induced by cells treated with glutaraldehyde or trypsin. In addition, hapten-specific B-cell unresponsiveness could not be induced by FITC-labelled glutaraldehyde-treated syngeneic lymphocytes. However, cold targets treated with trypsin or glutaraldehyde efficiently blocked T-cell-mediated cytotoxicity. The requirement for H-2-coded antigens for B-cell activation against haptenated syngeneic lymphocytes is discussed.